Vol. 60, Issue 4, 666-673, October 2001

Reciprocal Modulation of _2A-Adrenoceptor and G_o Protein States as Determined by Carboxy-Terminal Mutagenesis of a G_o Protein

Thierry Wurch, Junko Okuda,¹ and Petrus J. Pauwels

Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, Castres, France

The C-terminal portion of G proteins plays a key role in their selective activation by cognate receptors. _2A-Adrenoceptors (_2A-ARs) can differentially inhibit or stimulate adenylyl cyclases by the activation of distinct G_i/o and G_s protein families. The implication of the C-terminal portion of G_o and G_s proteins in their activation by _2A-ARs was analyzed by constructing mutant G_o proteins in which each of the last five amino acid positions were exchanged for those corresponding to a G_s protein. Agonist-dependent, pertussis toxin-resistant binding of guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) revealed that the degree of positive efficacy of clonidine was highly dependent on the presence of a G_o protein-derived Gly amino acid as the 3 residue at the C-terminal portion of the protein. In contrast, antagonist properties for clonidine were observed for those mutants carrying a G_s protein-derived Glu residue at this position. ()-Epinephrine yielded almost similar maximal [³⁵S]GTPS binding responses, but its potency was decreased 22- to 150-fold at the 3 Glu containing mutant G_o proteins compared with those mutants containing a Gly. A 9- to 39-fold increase in the _2A-AR agonist equilibrium dissociation constants further reflected changes in the G protein-induced _2A-AR state mediated by the specific Gly to Glu mutation in the C-terminal portion of the G_o protein. The present data emphasize the unique role of the 3 position at the G protein C-terminal portion, independent of its surrounding peptidic environment, in constraining a structure favorable for activated receptor interaction and transmission of the mutation-induced conformational change from the G_o protein to the _2A-AR.